Multiple responses optimization of antioxidative components extracted from Fenugreek seeds using response surface methodology to identify their chemical compositions

Abstract Fenugreek seeds (Trigonella foenum‐graecum L.), one kind of traditional Chinese medicine, are reported to be of great potential as a new alternative in terms of their bioactive components. In our present study, an ultrasonic‐assisted method was applied in the extraction of antioxidative components from fenugreek seeds. Four factors: ethanol concentration, liquid–solid ratio, sonication time, and sonication power were selected and multiple responses were studied using the response surface methodology (RSM). The effects of factors along with the correlation between all responses (flavonoids content, 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) assay, OH− assay) were studied. The regression model indicated that all four factors are of significant effect on all responses. The model predicted that the ethanol concentration of 72%, solvent‐to‐material ratio of 35 ml/g, ultrasonic time of 41 min, and 500 W of power would provide a flavonoid yield of 9.10 mg/g, DPPH clearance of 80.33%, and OH− clearance of 24.28%, respectively. The confirmation test showed the closeness of the predicted results with those of experimental values. And AB‐8 resin was successfully used to purify the fenuellus hulusi seed extract, and the flavonoid concentration of 78.14% was obtained. Six flavonoids (Swertisin, Puerarin apioside, Jasminoside B, Astragalin, Apigenin‐7‐O‐beta‐D‐glucoside, and Apiin) were successfully identified by the liquid chromatography–mass spectrometry (LC–MS) analysis.


| INTRODUC TI ON
Flavonoids are secondary metabolites that are ubiquitous in plants.
Modern research shows that flavonoids are a common natural antioxidant. Their unique chemical structure makes them easy to be oxidized, thus showing strong in vivo antioxidant capacity. Studies have confirmed that the flavonoid Flavokawain B can protect cells from hydrogen peroxide (H 2 O 2 ) damage by neutralizing reactive oxygen species (ROS) in the cells (Yeap et al., 2017). The study of Martin et al. found that 8-prenylnaringenin has a strong antioxidant activity, showing the ability to protect colonic epithelial cells from oxidative stress damage (Ambrož et al., 2019). Upadhyay et al. found that 5-O-demethylnobiletin obtained from Okinawa plant extracts had good 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging ability (Upadhyay et al., 2014), and the scavenging ability of the flavonoids to DPPH free radical showed a significant positive correlation with the concentration of the compound. Due to the various biological activities of flavonoids, they have received extensive attention in the field of health in recent years. Research has confirmed that free radicals are closely related to human cardiovascular disease, physiological aging, and other factors that threaten public health. They have a strong oxidizing property, which can cause oxidative stress in people and endanger their health (Furukawa et al., 2004;Mohammad-Sadeghipour et al., 2020). Butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) are currently the most common synthetic antioxidants (Topal et al., 2015;Zhang et al., 2015). They are often used as food additives to prevent or delay food oxidation, improve food stability, and prolong the shelf life. However, toxicological experiments have shown that these synthetic antioxidants have relatively large toxic and side effects (Girgih, 2014), and have adverse effects on the human liver, spleen, and lungs. And antioxidant experiments also show that these commonly used synthetic antioxidants have weaker antioxidant effects than natural extracts. Side effects of synthetic antioxidants make it necessary to find a natural alternative.
Fenugreek seeds are internationally recognized as the most efficient edible and medicinal plant in the field of biological antioxidants (Kaviarasan et al., 2007). Modern pharmacological studies have shown that fenugreek seeds are a potential natural antioxidant plant (Madhava Naidu et al., 2011). Total flavonoids from fenugreek seeds can scavenge hydroxyl radicals (OH-) and inhibit H 2 O 2 -induced mitochondrial lipid peroxidation in rat hepatocytes. Trigonelline can inhibit the formation of tumor necrosis factor alpha (TNFα), cause hemoglobin glycation and lipid accumulation, and downregulate the gene expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and mitochondrial electron transport system (Tharaheswari et al., 2014;Prabhjot et al., 2021), and inhibit or slow down the oxidative stress of the body. In addition, fenugreek seed extract also has lipid, hypoglycemic, hypolipidemic, and cardiovascular disease related links with its antioxidant capacity (Omri et al., 2019;Sur et al., 2010;Khole et al., 2014;Reddy & Srinivasan, 2009). The flavonoids in fenugreek seeds, in particular, are the main source of fenugreek's antioxidant power. Therefore, the total flavonoids of fenugreek seeds have a high research value in an antioxidant.
Considering the high antioxidative potential of fenugreek seeds, extraction optimization of bioactive components is our priority.
Herein, a multiple responses surface methodology (MRSM) was applied to optimize the extraction of fenugreek seed. The effects of factors along with the correlation between all responses (flavonoids content, DPPH assay, OH − assay) were studied.

| Materials
Fenugreek seeds were obtained from Anhui Dechang Pharmaceutical Co., Ltd. Dried seeds were ground into powder and then screened with a 60-mesh sieve. Fenugreek seed powder was extracted by a Soxhlet extractor with petroleum ether (60-90°C) as the extractant, and after 30 min, petroleum ether was filtered out to obtain fenugreek seed powder. The powder was placed in a plastic bag and stored in the refrigerator at 4°C before use. All other chemicals and solvents (analytical and high-performance liquid chromatography [HPLC] grades) were purchased from Shanghai Sinopharm Group Co., Ltd.

| Ultrasound-assisted extraction (UAE)
An ultrasonic processor (500 Watts and 40 kHz model, Yongkang Ultrasonic Co., Ltd.) was used. The extraction was performed using ethanol/water as a solvent in different proportions with a total extraction volume of 100 ml. After each extraction, the extracts were vacuum filtered on Whatman filter paper No. 4, and the filtrates stored at −18°C ± 2°C until analysis.

| Total flavonoids content (TFC)
The total flavonoid in fenugreek seed extract was measured according to the technique described by the sodium nitrite-sodium nitrate aluminum hydroxide colorimetric method. Rutin was used to give the standard curve: y = 12.54x−0.0104 and R 2 = .9979, where y is the absorbance value of the sample and x is the sample concentration.
TFC was expressed as the rutin equivalent (RE) per gram of FPFS (freeze-drying powders of Fenugreek seeds) (milligrams (mg) RE/g) and calculated by Equation (1).

| Antioxidant activities
All antioxidative effects were determined by using techniques which have already been reported.
The DPPH clearance rate could be calculated as follows: The OH removal rate could be calculated as follows: A: 6 mmol ethanol-salicylic acid, 7.5 mmol ferrous sulfate solution, and 4% H 2 O 2 solution were mixed at a ratio of 1:1:1. A 0 : A mixture solution of A and distilled water. A 1 : A mixture solution of A and sample. A 2 : A mixture solution of distilled water and sample.

| Identification of antioxidant substances in extracts
After optimizing the extraction method, the Fenugreek seeds were extracted under the optimal conditions, and the extraction solution was configured as 0.5 mg/ml, and the pH was adjusted to 5. An AB-8 macroporous resin was used to adsorb the extract, and 3 bV (bed volumes) of 40% ethanol was used for elution after the adsorption.
The eluent was collected step by step in 5 ml units, and flavonoid content in the eluent was determined successively. The eluent with a higher flavonoid content was combined and analyzed by LC-MS (Samoticha et al., 2017).
Thermo Scientific Hypersil Gold (100 mm × 2.1 mm, 1.9 μm) was selected as the chromatographic column, and gradient elution was

| Single-factor experiments
Flavonoids were commonly considered as an important natural antioxidant in different herbs. Thus, the total flavonoids content (TFC) was selected as a quota in the present single-factor experiments.
Influences of different ethanol/water systems, solvent-to-material ratio, ultrasonic time, and ultrasonic power were investigated.
Results indicated that the extraction efficiency was affected by ethanol concentration, ultrasonic time, power, and solvent-to-material ratio ( Figure 1).
When the ethanol concentration increased from 15% to 60%, the TFC increased significantly, and after more than 60%, the extraction rate began to decline, indicating that the extraction rate of total flavonoids from fenugreek seeds was the highest at this concentration ( Figure 1a); solvent-to-material ratio is one of the factors affecting TFC (Figure 1b). When the liquid-to-solid ratio increases from 10 ml/g to 20 ml/g, TFC increases rapidly. At 25 ml/g, TFC grows slowly, and when the liquid-to-material ratio exceeds 25 ml/g, its concentration tends to increase, so the contact area of solvent and solid reaches saturation with the increase of solvent-to-material ratio; That is the reason for the high extraction rate of flavonoids.

| Optimizing extraction conditions by the Box-Behnken design
Response surface methodology (RSM) was performed to optimize the extraction conditions using Design-Expert Ver. 8.1.5 (Stat-Ease Inc.). The Box-Behnken design (BBD) was used to find the optimal values for four independent variables: ethanol concentration, The flavonoids content of extracts (mg RE) weight of FPFS (g) ultrasonic time, power, and solvent-to-material ratio at three different levels (−1, 0, +1) ( Table 1).
The interactions between these four main factors could be obtained by this experimental design. The entire study comprised 29 separate experiments and each treatment was tested in triplicate.  (Norziah & Fezea, 2015).

As shown in
The regression coefficients of the quadratic predicted model are indicated in Table 3.
The quadratic polynomial equation was fitted by response surface regression as follows: where Y is the response variable; X i and X j are independent variables; 0 is the constant coefficient; i is the linear coefficient; ii is the quadratic coefficient; and ij is the cross-product coefficient.
The DPPH-RSC (radical scavenging activity) is mainly related to X 2 X 3 , followed by X 2 X 3 X 1 X 3 , X 3 , X 1 X 4 , X 4 , X 2 . Table 3 shows that the F value of model Y DPPH-RSC is 9.84 with a p-value <.0001, indicating that the model is significant and can be used for subsequent optimization designs. Moreover, the p-values of X 1 X 4 , X 4 , X 2 are lower than .01; thus, these factors significantly impact Y DPPH-RSC . The pvalues of X 2 X 3 X 1 X 3 , X 3 are lower than .001, showing that the influence of ethanol concentration and ultrasonic time on DPPH-RSC is exceptionally significant. OH-RSC is mainly related to X 1 , X 2 , followed by X 4 2 , X 1 X 4 . Table 3 shows that the model Y OH-RSC has an Fvalue of model Y OH-RSC of 7.44, with a p-value <.0001, indicating that the model is significant and can be used for subsequent optimization designs. These results also mean that X 4 2 , X 1 X 4 (p-value <.05) have a significant impact on OH-RSC. Specifically, the linear effect of X 1 , X 2 shows to be highly significant (p-value <.0001). Table 3 shows that the F value of model Y TFC is 25.83 with a p-value <.0001, indicating that the model is significant and can be used for subsequent optimization designs. Also, X 2 2 , X 4 values (p-value <.05) indicated a major influence on TFC. More specifically, the linear effect of ethanol concentration, ultrasonic time, and solvent-to-material ratio (X 1 , X 2 , X 3 ) implied a remarkably significant (p < .001) positive effect on TFC.

| Analysis of the response surface
It can be seen from Figure 2a that the interaction of X 1 and X 4 has a significant positive effect on TFC, and the interaction of ethanol concentration and ultrasonic power (X 1 X 4 ) has a significant (p < .05) positive effect on TFC. TFC was gradually increased at lower ethanol concentration and lower ultrasonic power. However, with the increase of ultrasonic power and ethanol concentration, the increase of TFC gradually flattened and started to decrease, which may be because TFC is a polar compound and is more soluble in solvents with similar polarities . As the concentration increases and decreases, the polarity of the extract also decreases and increases, thereby reducing the TFC. With the increase of ultrasonic power, the cell walls of fenugreek seeds are more easily broken, and it is easier to release flavonoids (He et al., 2016). When the ultrasonic power is too high, the compounds in the fenugreek will accumulate more bonds, resulting in the destruction of the compound structure. Therefore, with the continuous increase of ultrasonic power, TFC will show a downward trend. The solvent-tomaterial ratio and extraction time showed a significant negative effect on TFC. It can be seen from Figure 2b that with the increase of the solvent-to-material ratio, the TFC showed a continuous upward trend, and the increase of TFC was in the case of a longer extraction time and it will be more pronounced. This shows that increasing the liquid-solid ratio during extraction is very important to improve the TFC yield, because with the increase of the solvent amount, the diffusion pressure also increases, which promotes as many flavonoid molecules as possible to dissociate into the solution, and the extraction rate is correspondingly increased (Yan et al., 2011). With the improvement of cavitation, the particle collisions generated by F I G U R E 2 Response surface three-dimensional (3D) plots for the interaction effects of independent variables of ethanol concentration (%), solvent-to-material ratio (ml/g), extraction ultrasonic time (min), and ultrasonic power (W) on dependent variables of total flavonoids content (TFC) (a, b), 2-2-dipheny-1-picrylhydrazyl (DPPH) (c-e), and OH − (f)

TA B L E 4 Chemical compositions of extracts from
Fenugreek seeds cavitation can also destroy the cell wall better and promote the exudation of components (Zhang et al., 2019). In the case of a longer extraction time, flavonoids are also more likely to be precipitated, which makes the cavitation effect more obvious.
As can be seen in Figure 2c, the interaction of X 1 X 4 has a significant effect on DPPH-RSC, which is consistent with TFC. And the three-dimensional (3D) map also appears to be consistent with Possibly due to the increase of X 1 and the decrease of polarity, some flavonoids with a higher polarity could not be dissolved, and the yield of TFC and the scavenging rate of OH-radicals decreased. Under the same conditions, the DPPH clearance rate was increased, and the flavonoids with lower polarity may have a better effect on the DPPH clearance rate (Norlia et al., 2014;Yang et al., 2002). The interaction of ethanol concentration and sonication time had a significant effect on the DPPH clearance, which indirectly confirmed this speculation.

| Identification of compounds
The extract of Fenugreek seeds was purified by AB-8 resin and the flavonoid content of 71.84% was obtained. Six flavonoids, such as  Table 4, and the total ion current spectrum is shown in Figure 3.

| Model validation
A MRSM method was used to determine the optimal combination of each factor and level. The optimum extraction conditions were determined by Design-expert software. The optimal conditions for extracting TFC and its antioxidant activity were as follows: ethanol concentration of 72%, solvent-to-material ratio of 35 ml/g, ultrasonic time of 41 min, and ultrasonic power of 500 W.
According to the improved optimal process conditions, verification experiments were repeated three times; the results are shown in

| CON CLUS ION
Herein, an extraction method was applied to optimize the extraction of fenugreek seeds. The effects of factors along with the correlation between all responses (flavonoids content, DPPH assay, and OH − assay) were studied.
The medicinal value of total flavonoids in fenugreek seed obtained by ultrasound-assisted multiple responses surface methodology (MRSM) technology and their use as natural antioxidants were assessed in this study. According to a multiple response surface, the optimal extraction parameters of TFC yield and antioxidant activity of fenugreek under different conditions were determined, and the parameters were X 1 = 72%, X 2 = 35 ml/g, X 3 = 41 min, and X 4 = 500 W. Under these optimal conditions, the extraction flavonoid yield of 9.10 mg/g, DPPH clearance of 80.33%, and OH − clearance of 24.28%, respectively, were observed. In addition, the results showed that the ultrasonic-assisted multiresponse surface methodology was used to optimize the extraction of TFC yield and antioxidant activity was significantly improved. Flavonoids are thought to be a natural antioxidant that protects against many diseases caused by the oxidative stress. Overall, these results provide new insights into the extraction of flavonoids from fenugreek seeds and the study of antioxidant activity. And AB-8 resin was successfully used Note: X 1 : ethanol concentration (%); X 2 : solvent-to-material ratio (ml/g); X 3 : ultrasonic time (min); X 4 : ultrasonic power (W). The experimental results were all expressed as mean ± standard deviation (n = 3).

TA B L E 5 Experimental values and
predicted values of response variables at optimum extraction conditions to purify fenuellus hulusi seed extract, and the flavonoid concentration of 78.14% was obtained. Six flavonoids (Swertisin, Puerarin apioside, Jasminoside B, Astragalin, Apigenin-7-O-beta-D-glucoside, and Apiin) were successfully identified by the LC-MS analysis.

This research was supported by The Open Fund of State Key
Laboratory of Tea Plant Biology and Utilization (SKLTOF20200106) and the Anhui Provincial Major Scientific and Technological Special Project (Grant No. 17030701023).

CO N FLI C T O F I NTE R E S T
The authors declare that there are no conflicts in interest.

E TH I C A L A PPROVA L
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S TATE M E NT O F H U M A N A N D A N I M A L R I G HT S
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